This invention is related to cellular senescence and changes in cellular gene expression that accompany senescence. In particular, the invention is related to the identification of genes the expression of which is modulated by a cellular gene product, p21, induced in cells at the onset of senescence. More specifically, the invention provides markers of cellular senescence that are genes whose expression in induced or repressed by p21. The invention provides methods for identifying compounds that inhibit or potentiate cellular senescence by detecting inhibition of repression or induction of these marker genes. Also provided are reagents that are recombinant mammalian cells containing a recombinant expression construct encoding p21 that is experimentally-inducible, and recombinant mammalian cells containing a recombinant expression construct that expresses a reporter gene under the transcriptional control of a promoter for a gene that is regulated by p21.
p21WAF1/CIP1/SDI1 is an important mediator of growth arrest and senescence in mammalian cells. p21 has been independently identified by several groups as a protein that binds and inhibits cyclin-dependent kinases (CDK) (Harper et al., 1993, Cell 75: 805-816), as a gene upregulated by wild-type p53 (el-Deiry et al., 1993, Cancer Res. 55: 2910-2919), and as a growth-inhibitory gene overexpressed in senescent fibroblasts (Noda et al., 1994, Exp. Cell. Res. 211: 90-98). Because of its pivotal role in p53-regulated growth arrest, p21 is usually regarded as a tumor suppressor. Nevertheless, p21 mutations in human cancer are rare (Hall and Peters, 1996, Adv. Cancer Res. 68: 67-108), and p21 knockout mice develop normally and do not show an increased rate of tumorigenesis (Deng et al., 1995, Cell 82: 675-684).
Cellular levels of p21 are increased in response to a variety of stimuli, including DNA-damaging and differentiating agents. Some of these responses are mediated through transcriptional activation of the p21 gene by p53, but p21 is also regulated by a variety of p53-independent factors (reviewed in Gartel and Tyner, 1999, Exp. Cell Res. 227: 171-181). Increased p21 expression leads to cell growth arrest (Noda et al., 1994, ibid.), which occurs in both G1 and G2 (Niculescu et al., 1998, Mol. Cell. Biol. 18: 629-643) and is accompanied by the development of morphologic and phenotypic markers of senescence (Vogt et al., 1998, Cell Growth Differ. 9: 139-146; McConnell et al., 1998, Curr. Biol. 8: 351-354; Bates et al., 1998, Oncogene 17: 1691-1703; Fang et al., 1999, Oncogene 18: 2789-2797).
Transient induction of p21 mediates different forms of damage-induced growth arrest, including transient arrest that allows cell to repair DNA damage, as well as permanent growth arrest (also termed xe2x80x9caccelerated senescencexe2x80x9d), which is induced in normal fibroblasts (DiLeonardo et al., 1994, Genes Develop. 8: 2540-2551; Robles and Adami, 1998, Oncogene 16: 1113-1123) and tumor cells (Chang et al., 1999, Cancer Res. 59: 3761-3767) by DNA damage or introduction of oncogenic RAS (Serrano et al., 1997, Cell 88: 593-602). A surge of p21 expression also coincides with the onset of terminal growth arrest during replicative senescence of aging fibroblasts (Noda et al., 1994, ibid.; Alcorta et al., 1996, Proc. Natl. Acad. Sci USA 93:13742-13747; Stein et al., 1999, Mol. Cell. Biol. 19: 2109-2117) and terminal differentiation of postmitotic cells (El-Deiry et al., 1995, ibid.; Gartel et al., 1996, Exp. Cell Res. 246: 280-289). Analysis of cells that cannot express p21 (p21xe2x88x92/xe2x88x92homozygotes) demonstrated the requirement of p21 in transient G1 and G2 arrest (Deng et al., 1995, ibid.; Waldman et al., 1995, Cancer Res. 55: 5187-5190; Bunz et al., 1998, Science 282: 1497-1501), in replicative senescence of normal fibroblasts (Brown et al., 1997, Science 277: 831-834), and in accelerated senescence of tumor cells (Chang et al., 1999, Oncogene 18: 4808-4818).
While p21 is not a transcription factor per se, it has indirect effects on gene expression that may play a role in its cellular functions. The best-known biochemical function of p21 is the inhibition of CDK complexes that regulate transitions between different phases of the cell cycle (reviewed in Cartel and Tyner, 1998, xe2x80x9cThe growth-regulatory role of p21 (WAF1/CIP1),xe2x80x9d in Inhibitors of Cell Growth, Progess in Molecular and Subcellular Biology, Vol. 20 (A. Macieir-Coelho, ed.), Springer-Verlag: Berlin Heidelberg, pp. 43-71.). One of the consequences of CDK inhibition is dephosphorylation of Rb, which in turn inhibits E2F transcription factors that regulate many genes involved in DNA replication and cell cycle progression (Nevins, 1998, Cell Growth Differ. 9: 585-593). A comparison of p21-expressing cells (p21+/+) and p21-nonexpressing cells (p21xe2x88x92/xe2x88x92) has implicated p21 in radiation-induced inhibition of several E2F-regulated cellular genes (de Toledo et al., 1998, Cell Growth Differ. 9: 887-896). Another result of CDK inhibition by p21 is stimulation of transcription cofactor p300 that augments NFxcexaB (Perkins et al., 1988, Science 275: 523-527). Activation of histone acetyltransferase p300, that enhances many inducible transcription factors, may have a pleiotropic effect on gene expression (Snowden and Perkins, 1988, Biochem. Pharmacol. 55: 1947-1954). p21 may also affect gene expression through its interactions with proteins other than CDK. For example, p21 has been found to inhibit the expression of keratinocyte differentiation markers; this effect was dependent on the C-terminal portion of p21, which is not required for CDK inhibition but is known to bind the proliferating cell nuclear antigen (Di Cunto et al., 1998, Science 280: 1069-1072). p21 was also reported to bind JNK kinases (Shim et al., 1996, Nature 381: 804-807), apoptosis signal-regulating kinase 1 (Asada et al., 1999, EMBO J. 18: 1223-1234), and Gadd45 (Kearsey et al., 1995, Oncogene 11: 1675-1683); these interactions may affect the expression of genes regulated by the corresponding pathways.
There remains a need in this art to identify genes whose expression is modulated by induction of p21 gene expression. There is also a need in this art to develop targets for assessing the effects of compounds on cellular senescence, carcinogenesis and age-related diseases.
This invention provides reagents and methods for identifying genes whose expression is modulated by induction of p21 gene expression. The invention also provides reagents and methods for identifying compounds that inhibit or potentiate the effects of p21 on cellular gene expression, as a first step in rational drug design for preventing cellular senescence, carcinogenesis and age-related diseases or for increasing the efficacy of anticancer therapies.
In a first aspect, the invention provides a mammalian cell containing an inducible p21 gene. In preferred embodiments, the mammalian cell is a recombinant mammalian cell comprising a recombinant expression construct encoding an inducible p21 gene. More preferably, the construct comprises a nucleotide sequence encoding p21, most preferably human p21, under the transcriptional control of an inducible promoter. In alternative embodiments, the construct comprises a nucleotide sequence encoding the amino-terminal portion of p21 comprising the CDK binding domain, more preferably comprising amino acids 1 through 78 of the p21 amino acid sequence. In more preferred embodiments, the inducible promoter can be induced by contacting the cells with an inducing agent, most preferably a physiologically-neutral inducing agent, that induces transcription from the inducible promoter or by removing an agent that inhibits transcription from such promoter. In a preferred embodiment, the mammalian cell is a fibrosarcoma cell.
In another embodiment of the first aspect of the invention are provided recombinant mammalian cells comprising a recombinant expression construct in which a reporter gene is under the transcriptional control of a promoter derived from a cellular gene whose expression is modulated by p21. In a preferred embodiment, the promoter is derived from a cellular gene whose expression is repressed by p21. In these embodiments, the promoter is most preferably derived from a gene identified in Table I. Most preferably, the promoter is derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin xcex1, MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA polymerase xcex1, Rad54, exonuclease HEX1/RAD2, or citron kinase. In other preferred embodiments, the promoter is derived from a cellular gene whose expression is induced by p21. In these embodiments, the promoter is most preferably derived from a gene identified in Table II. Most preferably, the promoter is derived from serum amyloid A, complement C3, connective tissue growth factor, integrin xcex2-3, activin A, natural killer cell protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide dismutase 2, or cathepsin B. Preferred reporter genes comprising the recombinant expression constructs of the invention include firefly luciferase, chloramphenicol acetyltransferase, beta-galactosidase, green fluorescent protein, or alkaline phosphatase.
In additional preferred embodiments, the invention provides a mammalian cell comprising a first recombinant expression construct encoding a reporter gene under the transcriptional control of a promoter for a mammalian gene whose expression is modulated by p21, and a second recombinant expression construct encoding a mammalian p21 gene, wherein expression of p21 is experimentally-induced in the mammalian cell thereby. In preferred embodiments, the recombinant expression construct encoding a mammalian p21 gene is under the transcriptional control of an inducible heterologous promoter, wherein expression of p21 from the recombinant expression construct is mediated by contacting the recombinant cell with an inducing agent that induces transcription from the inducible promoter or by removing an agent that inhibits transcription from such promoter. Preferably, the construct comprises a nucleotide sequence encoding p21, most preferably human p21. In alternative embodiments, the construct comprises a nucleotide sequence encoding the amino-terminal portion of p21 comprising the CDK binding domain, more preferably comprising amino acids 1 through 78 of the p21 amino acid sequence. In a preferred embodiment, the promoter is derived from a cellular gene whose expression is repressed by p21. In these embodiments, the promoter is most preferably derived from a gene identified in Table I. In other preferred embodiments, the promoter is derived from a cellular gene whose expression is induced by p21. In these embodiments, the promoter is most preferably derived from a gene identified in Table II. Preferred reporter genes comprising the recombinant expression constructs of the invention include firefly luciferase, chloramphenicol acetyltransferase, beta-galactosidase, green fluorescent protein, or alkaline phosphatase. In a preferred embodiment, the mammalian cell is a fibrosarcoma cell.
In a second aspect, the invention provides a conditioned cell culture medium wherein the medium is conditioned by cells expressing p21. A method for producing said conditioned medium, comprising the step of culturing p21-expressing cells in a mammalian cell culture medium is also provided.
In a third aspect, the invention provides methods for identifying compounds that inhibit p21-mediated modulation of cellular gene expression. These methods comprise the steps of inducing or otherwise producing p21 in a mammalian cell; assaying the cell in the presence of the compound for changes in expression of cellular genes whose expression is modulated by p2l; and identifying compounds that inhibit p21-mediated modulation of cellular gene expression if expression of the cellular genes is changed to a lesser extent in the presence of the compound than in the absence of the compound. In preferred embodiments, the cellular genes are repressed by p21, and inhibitors are detected by detecting expression of the genes at levels greater than those detected when p21 is expressed in the absence of the compound. In preferred embodiments, the genes are identified in Table I. In alternative preferred embodiments, the cellular genes are induced by p21, and inhibitors are detected by detecting expression of the genes at levels less than those detected when p21 is expressed in the absence of the compound. In preferred embodiments, the genes are identified in Table II. In further alternative embodiments, the method is performed using a recombinant mammalian cell comprising a reporter gene under the transcriptional control of a promoter derived from a gene whose expression is modulated by p21. In these embodiments using constructs comprising promoters derived from genes repressed by p21, the reporter gene product is produced at greater levels in the presence than in the absence of the compound when the compound is an inhibitor of p21 gene expression modulation. In these embodiments, the promoter is most preferably derived from a gene identified in Table I. Most preferably, the promoter is derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin xcex1, MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA polymerase xcex1, Rad54, exonuclease HEX1/RAD2, or citron kinase. When using constructs comprising promoters derived from genes induced by p21, the reporter gene product is produced at lesser levels in the presence than the absence of the compound when the compound is an inhibitor of p21 gene expression modulation. In these embodiments, the promoter is most preferably derived from a gene identified in Table II. Most preferably, the promoter is derived from serum amyloid A, complement C3, connective tissue growth factor, integrin xcex2-3, activin A, natural killer cell protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide dismutase 2, or cathepsin B. Preferred reporter genes comprising the recombinant expression constructs of the invention include firefly luciferase, chloramphenicol acetyltransferase, beta-galactosidase, green fluorescent protein, or alkaline phosphatase. In other preferred embodiments, the cell comprises a first recombinant expression construct encoding a reporter gene under the transcriptional control of a promoter for a mammalian gene whose expression is modulated by p21, and a second recombinant expression construct encoding a mammalian p21 gene, wherein expression of p21 is experimentally-induced in the mammalian cell thereby. The product of the reporter gene or the endogenous gene that is induced or repressed by p21 is detected using an immunological reagent, by assaying for an activity of the gene product, or by hybridization to a complementary nucleic acid.
In a fourth aspect, the invention provides methods for identifying compounds that inhibit senescence in a mammalian cell. These methods comprise the steps of treating the mammalian cell in the presence of the compound with an agent or culturing the mammalian cell under conditions that induce senescence; assaying the mammalian cell for repression or induction of genes that are repressed or induced by p21 gene expression; and identifying the compound as an inhibitor of senescence if genes that are repressed by p21 are not repressed, or genes that are induced by p21 are not induced, in the presence of the compound. In preferred embodiments, the cellular genes are repressed by p21, and senescence inhibitors are identified by detecting expression of the genes at levels greater than those detected when p21 is expressed in the absence of the compound. In preferred embodiments, the genes are identified in Table I. In alternative preferred embodiments, the cellular genes are induced by p21, and senescence inhibitors are detected by detecting expression of the genes at levels less than those detected when p21 is expressed in the absence of the compound. In preferred embodiments, the genes are identified in Table II. In further alternative embodiments, the method is performed using a recombinant mammalian cell comprising a reporter gene under the transcriptional control of a promoter derived from a gene whose expression is modulated by p21. In these embodiments, production of the product of the reporter gene at greater levels in the presence than in the absence of the compound when using constructs comprising promoters derived from genes repressed by p21, or at lesser levels in the presence than the absence of the compound when using constructs comprising promoter derived from genes induced by p21, is detected when the compound is an inhibitor of senescence. The promoters are preferably derived from genes identified in Table I (for genes repressed by p21) or Table II (for genes induced by p21). For p21-repressed IS genes, the promoter is most preferably derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin xcex1, MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA polymerase xcex1, Rad54, exonuclease HEX1/RAD2, or citron kinase. For p21-induced genes, the promoter most preferably is derived from serum amyloid A, complement C3, connective tissue growth factor, integrin xcex2-3, activin A, natural killer cell protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide dismutase 2, or cathepsin B. In other preferred embodiments, the cell comprises a first recombinant expression construct encoding a reporter gene under the transcriptional control of a promoter for a mammalian gene whose expression is modulated by p21, and a second recombinant expression construct encoding a mammalian p21 gene, wherein expression of p21 is experimentally-induced in the mammalian cell thereby. The product of the reporter gene or the endogenous gene that is induced or repressed by p21 is detected using an immunological reagent, by assaying for an activity of the gene product, or by hybridization to a complementary nucleic acid.
In a fifth aspect, the invention provides methods for inhibiting cellular senescence, age-related diseases or age-associated gene products, the method comprising the steps of contacting the cell with a compound that inhibits senescence as determined using the methods provided in the aforesaid aspects of the invention.
In a sixth aspect, the invention provides methods for identifying compounds that potentiate senescence in a mammalian cell. These methods comprise the steps of inducing p21 in the mammalian cell in the presence and absence of the compound; assaying the mammalian tumor cell for repression or induction of genes that are repressed or induced by p21 gene expression; and identifying the compound as a potentiator of senescence if genes that are repressed by p21 are repressed to a greater extent, or genes that are induced by p21 are induced to a greater extent, in the presence of the compound. In preferred embodiments, the cellular genes are repressed by p21, and potentiators are detected by detecting expression of the cellular gene at levels less than those detected when p21 is expressed in the absence of the compound. In preferred embodiments, the genes are identified in Table I. In alternative preferred embodiments, the cellular genes are induced by p21, and potentiators are detected by detecting expression of the cellular gene at levels greater than those detected when p21 is expressed in the absence of the compound. In preferred embodiments, the genes are identified in Table II. In further alternative embodiments, the method is performed using recombinant mammalian cells comprising a reporter gene under the transcriptional control of a promoter derived from a gene whose expression is modulated by p21, wherein the cells comprise constructs having the reporter gene under the transcriptional control of promoters from genes whose expression is modulated by p21. In these embodiments, production of the product of the reporter gene at lower levels in the presence than in the absence of the compound when using constructs comprising promoters derived from genes repressed by p21, or at greater levels in the presence than the absence of the compound when using constructs comprising promoter derived from genes induced by p21, is detected when the compound is a potentiator of senescence. In preferred embodiments, the promoters are derived from genes whose expression is repressed by p21, most preferably genes identified in Table I. Most preferably, the promoter is derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin xcex1, MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA polymerase xcex1, Rad54, exonuclease HEX1/RAD2, or citron kinase. In alternative preferred embodiments, the promoters are derived from genes whose expression is induced by p21, most preferably genes identified in Table II. Most preferably, the promoter is derived from serum amyloid A, complement C3, connective tissue growth factor, integrin xcex2-3, activin A, natural killer cell protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide dismutase 2, or cathepsin B. In other preferred embodiments, the cell comprises a first recombinant expression construct encoding a reporter gene under the transcriptional control of a promoter for a mammalian gene whose expression is modulated by p21, and a second recombinant expression construct encoding a mammalian p21 gene, wherein expression of p21 is experimentally-induced in the mammalian cell thereby. The product of the reporter gene or the endogenous gene that is induced or repressed by p21 is detected using an immunological reagent, by assaying for an activity of the gene product, or by hybridization to a complementary nucleic acid.
In a seventh aspect, the invention provides methods for promoting or potentiating cellular senescence in tumor cells, hyperplastic cells or any cell type that is pathological or disease-causing due to excessive proliferation, the method comprising the steps of contacting the cell with a compound that potentiates senescence as determined using the methods provided in the aforesaid aspect of the invention.
In a eighth aspect, the invention provides compounds that are identified using any of the methods of the invention as disclosed herein.
In a ninth aspect, the invention provides methods for obtaining a plurality of nucleic acid species enriched for genes involved in cell cycle progression. These methods comprise the steps of inducing the expression of p21 in a mammalian cell; obtaining cellular mRNA from a mammalian cell before p21 induction and after p21 is induced and cell growth is stopped; and obtaining the plurality of nucleic acid species enriched for genes involved in cell cycle progression. In a preferred embodiment, the plurality of nucleic acid species enriched for cell cycle progression genes is obtained by subtractive hybridization methods known in the art, whereby nucleic acid species underrepresented in cells expressing p21 are selectively enriched.
In a tenth aspect, the invention provides methods for obtaining a plurality of nucleic acid species enriched for genes that encode secreted proteins with paracrine functions and proteins involved in senescence and age-related diseases. These methods comprise the steps of inducing expression of p21 in a mammalian cell; obtaining cellular mRNA from a mammalian cell before and after p21 is induced; and obtaining the plurality of nucleic acid species enriched for genes whose expression is increased in the cell after p21 is induced. In preferred embodiments, the paracrine functions of the proteins are mitogenic and anti-apoptotic effects. In a preferred embodiment, the plurality of nucleic acid species enriched for genes that encode secreted proteins with paracrine functions and proteins involved in senescence and age-related diseases is obtained by subtractive hybridization methods known in the art, whereby nucleic acid species overrepresented in cells expressing p21 are selectively enriched.
In an eleventh aspect, the invention provides a method for identifying genes that are markers of cellular senescence, the method comprising the steps of inducing senescence by producing p21 expression in a first population of mammalian cells and inducing quiescence in a second population of mammalian cells; obtaining mRNA from each population of cells; comparing the pattern of gene expression in cells before and after production or p21 in the cells with the pattern of gene expression in cells before and after the cells became quiescent; comparing the plurality of genes strongly induced in the cells after p21 is produced with the plurality of genes strongly induced in quiescent cells; and identifying the genes strongly induced in cells producing p21 that are not strongly induced in quiescent cells.
In a twelfth aspect, the invention provides methods for detecting senescence in a mammalian cell. These methods comprise the step of detecting expression of a gene that is a marker for senescence. In preferred embodiments, preferred markers of senescence include connective tissue growth factor (CTGF), serum amyloid A, integrin xcex2-3, activin A, natural killer cell protein 4, Mac2 binding protein, or tissue transglutaminase.
In a thirteenth aspect, the invention provides methods for identifying compounds that promote induction of senescence in a mammalian cell. These methods comprise the steps of treating the mammalian cell with an agent or culturing the mammalian cell under conditions that induce senescence in the presence of the compound; assaying the mammalian tumor cell for repression or induction of genes that are repressed or induced by p21 gene expression; and identifying the compound as a potentiator of senescence if genes that are repressed by p21 are further repressed, i.e., to a greater extent, or genes that are induced by p21 are further induced, i.e., to a greater extent, in the presence of the compound. In preferred embodiments, the cellular genes are repressed by p21, and compounds that promote induction of senescence are detected by detecting expression of the cellular gene at levels less than those detected when p21 is expressed in the absence of the compound. In preferred embodiments, the genes are identified in Table I. In alternative preferred embodiments, the cellular genes are induced by p21, and compounds that promote induction of senescence are detected by detecting expression of the cellular gene at levels greater than those detected when p21 is expressed in the absence of the compound. In preferred embodiments, the genes are identified in Table II. In further alternative embodiments, the method is performed using recombinant mammalian cells comprising a reporter gene under the transcriptional control of a promoter derived from a gene whose expression is modulated by p21, wherein the cells comprise constructs having the reporter gene under the transcriptional control of promoters from genes whose expression is modulated by p21. In these embodiments, production of the product of the reporter gene at lower levels in the presence than in the absence of the compound when using constructs comprising promoters derived from genes repressed by p21, or at greater levels in the presence than the absence of the compound when using constructs comprising promoter derived from genes induced by p21, is detected when the compound promotes induction of senescence. In preferred embodiments, the promoters are derived from genes whose expression is repressed by p21, most preferably genes identified in Table I. Most preferably, the promoter is derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin xcex1, MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA polymerase xcex1, Rad54, exonuclease HEX1/RAD2, or citron kinase. In alternative preferred embodiments, the promoters are derived from genes whose expression is induced by p21, most preferably genes identified in Table II. Most preferably, the promoter is derived from serum amyloid A, complement C3, connective tissue growth factor, integrin xcex2-3, activin A, natural killer cell protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide dismutase 2, or cathepsin B. In other preferred embodiments, the cell comprises a first recombinant expression construct encoding a reporter gene under the transcriptional control of a promoter for a mammalian gene whose expression is modulated by p21, and a second recombinant expression construct encoding a mammalian p21 gene, wherein expression of p21 is experimentally-induced in the mammalian cell thereby. The product of the reporter gene or the endogenous gene that is induced or repressed by p21 is detected using an immunological reagent, by assaying for an activity of the gene product, or by hybridization to a complementary nucleic acid.
In a fourteenth aspect, the invention provides methods for identifying compounds that induce senescence in a mammalian cell. These methods comprise the steps of assaying a mammalian cell in the presence and absence of the compound for repression or induction of genes that are repressed or induced by p21 gene expression; and identifying compounds that induce senescence if genes that are repressed by p21 are repressed, or genes that are induced by p21 are induced, in the presence of the compound. In preferred embodiments, the cellular genes are repressed by p21, and compounds that induce senescence are detected by detecting expression of the cellular gene at levels less than those detected in the absence of the compound. In preferred embodiments, the genes are identified in Table I. In alternative preferred embodiments, the cellular genes are induced by p21, and compounds that induce senescence are detected by detecting expression of the cellular gene at levels greater than those detected in the absence of the compound. In preferred embodiments, the genes are identified in Table II. In further alternative embodiments, the method is performed using recombinant mammalian cells comprising a reporter gene under the transcriptional control of a promoter derived from a gene whose expression is modulated by p21, wherein the cells comprise constructs having the reporter gene under the transcriptional control of promoters from genes whose expression is modulated by p21. In these embodiments, production of the product of the reporter gene at lower levels in the presence than in the absence of the compound when using constructs comprising promoters derived from genes repressed by p21, or at greater levels in the presence than the absence of the compound when using constructs comprising promoter derived from genes induced by p21, is detected when the compound induces senescence. In preferred embodiments, the promoters are derived from genes whose expression is repressed by p21, most preferably genes identified in Table I. Most preferably, the promoter is derived from ORC1, PRC1, XRCC9, CDC2, cyclin B1, AIK1, CENP-A, CENP-F, MAD2, BUBR1, MCAK, HSET, CHL1, thymopoietin xcex1, MPP2, MPP5, CDC47/MCM7, CDC21/MCM4, DNA ligase I, DNA polymerase xcex1, Rad54, exonuclease HEX1/RAD2, or citron kinase. In alternative preferred embodiments, the promoters are derived from genes whose expression is induced by p21, most preferably genes identified in Table II. Most preferably, the promoter is derived from serum amyloid A, complement C3, connective tissue growth factor, integrin xcex2-3, activin A, natural killer cell protein 4, prosaposin, Mac2 binding protein, galectin-3, superoxide dismutase 2, or cathepsin B. The product of the reporter gene or the endogenous gene that is induced or repressed by p21 is detected using an immunological reagent, by assaying for an activity of the gene product, or by hybridization to a complementary nucleic acid.
Specific preferred embodiments of the present invention will become evident from the following more detailed description of certain preferred embodiments and the claims.